30 Jan

Dynamic Changes in Meiotic Progression: MATERIALS AND METHODS

Culture Media

All chemicals and reagents were from Sigma-Aldrich (Poole, U.K.) unless otherwise stated. The basic defined maturation medium (medium B) was Medium 199 containing Earle salts, 25 mM Hepes and sodium bicarbonate, 3 mM L-glutamine, 0.1% (w/v) BSA, 0.57 mM cysteine, 100 IU/ml of penicillin, and 0.1 mg/ml of streptomycin. The basal medium was supplemented with FSH (porcine, 50 ng/ml; National Institute of Diabetes and Digestive and Kidney Diseases [NIDDK]), LH (porcine, 0.2 |xg/ml; NIDDK), or EGF (human recombinant, 10 ng/ml) to formulate media F, L, and E, respectively, or supplemented in combinations to produce media LE, FL, FE, and FLE. The IVF medium was modified Tris-buffered medium (pH 9.9 at 4°C, balanced to pH 7.2 at 39°C in 5% CO2 for 12-24 h, containing 113 mM NaCl, 3 mM KCl, 7.5 mM CaCl2-2H2O, 20 mM Tris [crystallized free base], 11 mM glucose, and 5 mM sodium pyruvate) supplemented with 0.1% (w/v) BSA, 20 |xM adenosine (freshly prepared), 0.2 mM reduced glutathione (freshly prepared), and no antibiotics. The basic embryo IVC medium was NcSU 23 (pH 8.3 at 4°C, balanced to pH 7.4 at 39°C in 5% CO2 for 12-24 h, containing 108.7 mMNaCl, 4.8 mM KCl, 1.7 mM CaCl2 2H2O, 1.2 mM KH2PO4, 1.2 mM MgSO4-7H2O, 25.1 mM NaHCO3, 5.6 mM glucose, 1.0 mM glutamine, 7.0 mM taurine [freshly prepared], 5.0 mM hypotaurine [freshly prepared], and 0.4% [w/v] BSA) supplemented with 0.2 mM reduced glutathione (freshly prepared), 50 IU/ml of penicillin, and 50 |xg/ml of streptomycin. During the first 48 h of culture, glucose in the basic IVC medium was replaced with 4.5 mM sodium lactate (DL-lactic acid) and 0.3 mM sodium pyruvate. canada health and care mall

IVM of Oocytes and Assessment of Meiotic Stage

Pig ovaries were collected from a local commercial abattoir and transported to the laboratory in a warm flask in PBS (30-35°C) within 3 h of slaughter. Selected ovaries were washed three times in sterile PBS. Follicles (diameter, 3-5 mm) with a translucent appearance and extensive vascularization were aspirated using a 21-gauge needle attached to a 5/10-ml syringe primed with 0.5 ml of Dulbecco PBS (DPBS; Ca2+-free). The fluid was expelled into sterile Petri dishes (diameter, 5 cm) and held at 39°C while being inspected for oocytes. Cumulus-oocyte complexes (COCs), with more than three intact and compact cumulus layers were selected for culture after washing in DPBS supplemented with 0.1% polyvinyl alcohol (PVA) and in maturation media depending on the experiment. Groups of 25-50 COCs were cultured in single dishes (diameter, 35 mm; Nunc, Ros-kilde, Denmark) at a ratio of one oocyte per 10 ^l of maturation medium. To synchronize meiotic maturation, COCs first were preincubated in maturation medium supplemented with 5 |xg/ml of CHX for 12 h. The COCs then were thoroughly washed (three times in DPBS with 0.1% PVA and three times in maturation medium without CHX) and further cultured without CHX for various periods. All culture drops containing oocytes were covered with a thin layer of mineral oil pre-equilibrated with medium B and incubated in 5% CO2 in humidified air at 39°C.

To assess meiotic progression, single dishes of COCs were withdrawn from the incubator and the cumulus cells removed by vortexing for 2-3 min in medium containing 0.1% (w/v) hyaluronidase (type IV), 2 mM EDTA, 125 mM NaCl, 3 mM sodium citrate, and 10 mM Na2HPO4. Oocytes were mounted under coverslips on slides and fixed for 48 h with acetic acid:ethanol (1:3 v/v). After staining with 1% lacmoid in 45% acetic acid and destaining with acetoglycerol (20% acetic acid and 20% glycerol), the oocytes were examined under a phase-contrast microscope at 400 X magnification. Meiotic stages were classified as GV, GV breakdown (GVBD; diakinesis and prometaphase I), metaphase I (MI), anaphase I/ telophase I (AI/TI), and MII as described.

IVF, Assessment of Fertilization Parameters, and Embryo Culture

For sperm preparation, fresh extended pig semen (stored for up to 5 days at 21°C; PIC 225 semen, Pig Improvement Company, Oxford, U.K.) was washed twice by centrifugation (5 min, 500 X g) in DPBS (Ca2+-and Mg2+-free) supplemented with 0.1% BSA, 100 IU/ml of penicillin, and 100 |xg/ml of streptomycin. The sperm pellet was then resuspended in IVF medium and the sperm concentration determined by a hemocytom-eter after dilution in 18% NaCl saline. The sperm suspension (1 X 106 sperm cells/ml) was preincubated for a short period (10 min) at 39°C before coincubation with oocytes. At the end of maturation culture, dishes of oocytes were denuded of cumulus cells by brief vortexing in warm maturation medium (1-2 min), washed in the same medium, and transferred to IVF medium droplets, which were covered with mineral oil and preincubated for 2 h. Before insemination, oocytes were set in IVF-me-dium droplets (5-10 oocytes in 30 |xl of IVF medium; 4-7 drops per dish [diameter, 5 cm]) in the incubator for 20-30 min. An equal volume of sperm suspension (30 |xl) was introduced into each droplet, producing a final sperm concentration of 5 X 105 cells/ml. Oocyte-sperm coincubation was carried out for 6-8 h at 39°C under 5% CO2 in humidified air. After a brief wash in IVC medium, oocytes/putative zygotes were cultured (2550 in 600 |xl of IVC medium) for 2 days in four-well plates (Nunc). Cleaved embryos then were transferred into fresh IVC medium and cultured for a further 4 days. Putative zygotes and embryos were cultured at 39°C in humidified atmosphere of 5% CO2, 5% O2, and 90% N2. To assess fertilization parameters, oocytes/putative zygotes were fix-stained after IVF using the same procedure as that described above for assessment of the meiotic stage. Oocytes were considered to be penetrated when they had one or more (polyspermic) swollen sperm head(s) or male pronuclei with corresponding sperm tail(s).

Experiment 1

To determine the effects of FSH (F), LH (L), and EGF (E) on oocyte meiotic maturation and to compare the meiotic competencies of oocytes of different origin, oocytes collected from gilts and sows were cultured separately in the basic (B) and supplemented (F, L, E, LE, FL, FE, and FLE) maturation media. This was a full factorial experiment for three factors (FSH, LH, and EGF), each with two levels (presence or absence). At 44-48 h, all oocytes were fixed for examination of meiotic morphology. Four replicate dishes were used for each treatment combination, with a total of 1746 oocytes examined.

Experiment 2

To determine precisely the effects of hormones on the progression of meiotic maturation, oocytes of gilts and sows were synchronized by preincubation for 12 h with 5 |xg/ml of CHX in the basic (B) or supplemented (F, L, E, LE, FL, FE, and FLE) maturation media. Synchronized oocytes then were cultured in the same media without CHX for up to 36 h. At 12, 24, and 36 h, oocytes were fixed for examination of meiotic morphology. Three replicate dishes were used for each treatment at each time point, with a total of 2113 oocytes examined.

Experiment 3

To determine the effects of CHX pretreatment and FSH on oocyte development after fertilization, half of each batch of oocytes were synchronized with CHX for 12 h as in experiment 2 and then matured in medium LE for 26-28 h or FLE for 36 h. The other half were matured conventionally in LE medium for 40-42 h or in FLE medium for 48 h. Culture timings were coordinated so that both sets of oocytes were harvested simultaneously for IVF with the same sperm preparation. The proportions of cleaved oocytes/zygotes were recorded 2 days after IVF. Cleaving embryos were cultured for a further 4 days, and the blastocyst formation rates were recorded. Some matured oocytes from all treatments were overmatured or treated with sperm-free IVF procedures for at least 2 days. Three replicates were performed for each treatment, with a total of 506 oocytes cultured and fertilized.

A number of oocytes matured with or without CHX pretreatment were fertilized using the IVF procedure described above or after various modifications (e.g., to sperm concentration, number of oocytes in fertilization droplet, IVF medium). They were examined for polyspermic fertilization after coincubation with sperm for various periods of 6-22 h. In total, 349 oocytes/zygotes were examined.

Statistical Analysis

The proportions of meiotically responding (maturing or matured) oocytes, fertilized oocytes, and developing embryos out of the known total numbers of oocytes cultured in single wells were analyzed by fitting a generalized linear model assuming binomial errors. The significances of the factors of FSH, LH, EGF, and CHX and their interactions were tested using an analysis-of-deviance table (GenStat 6.1, VSN International, Oxford, U.K.). Analyzed data are presented as mean proportions and approximate SEMs predicted from the fitted model. A probability of P < 0.05 was considered to be statistically significant.

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